Our objective is to determine the effects of aging on types and rates of spontaneous genetic alterations in a mammalian system. Specifically, we will amplify and isolate individual mitochondrial DNA molecules from the liver of genetically defined Holstein cows by recombinant DNA techniques, sequence and compare limited portions of three functional genome regions: 1) a transcriptional processing site and 2) a region encoding the 3'-terminus of the large ribosomal RNA, as well as a relatively variable region within the mitochondrial D-loop. To calculate rates of nucleotide variation in these animals, we will compare cloned sequences within an animal, and as a function of age, tissue type, and maternal ancestry. Molecules cloned from periodic liver biopsy of the same animal will allow quantitation of mutations occurring during its lifespan. Such somatic mutation rates may be of fundamental importance in assessing the role of spontaneous genetic alterations in aging.